Dynamin-2 reduction rescues the skeletal myopathy of a SPEG-deficient mouse model

Figures

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  Figure 1. DNM2 expression and distribution in skeletal muscle from Speg-KO and WT mice. (A) SPEG-β and DNM2 coimmunoprecipitated from differentiated C2C12 myotube lysates with the use of rabbit anti-SPEG generated against a FLAG-tagged aortic preferentially expressed gene-1 fusion protein and anti-DNM2 antibodies. (B) Western blot analysis for DNM2 protein in Speg-KO skeletal muscles. Left panel shows representative image of DNM2 protein in Speg-KO versus WT quadriceps. Tubulin is used as a loading control. Right panel represents quantification of DNM2 expression relative to the expression of tubulin. Speg-KO mice demonstrated an average of 1.7-fold increase in DNM2 expression over WT in quadriceps and gastrocnemius muscles (**P ˂ 0.01, n = 7 per group; unpaired 2-tailed t test). (C) Immunostaining for DNM2 protein in tibialis anterior (TA) muscle from Speg-KO and WT mice (over 100 fibers were analyzed from each group, n = 3 per group). Speg-KO mice displayed an abnormal DNM2 accumulation (denoted by asterisks). Scale bars: 100 μm.

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  Figure 2. Generation of Speg-KO mice with DNM2 haploinsufficiency. (A) Representative images of control, Speg-KO, and Speg-rescue mice at 3 months of age and agarose gel analysis of DNA isolated from tails of mice showing the presence or absence of MCK-Cre (top), floxed Speg (middle), and DNM2 (bottom). MCK-Cre+ mice displayed bands for the transgene (~450 bp) and internal positive control (200 bp) while the floxed Speg allele is 485 bp in size versus 422 bp for the WT. DNM2-heterozygous mice displayed 2 bands at 553 bp and 1432 bp versus 1 band at 1432 bp for the WT. Ctrl, control; KO, Speg-KO; Res, Speg-rescue. (B) Immunoblot analysis and quantification of DNM2 in various types of striated muscles, including gastrocnemius (gastroc), triceps, diaphragm, and heart. (*P ˂ 0.05; **P ˂ 0.01, ***P ˂ 0.001, ****P < 0.0001, n = 4 per genotype; 1-way ANOVA with Tukey’s post hoc test.)

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  Figure 3. DNM2 reduction increases the life span and whole-body weight in Speg-rescue mice. (A) Life span of all Speg-rescue, Speg-KO, and control mice was monitored until 48 weeks of age. Data were represented as percentage survival (control, n = 35; Speg-KO, n = 26; Speg-rescue, n = 35). Survival rate for male (B) and female mice (C) were shown separately (for males: control, n = 21; Speg-KO, n = 12; Speg-rescue, n = 21; for females: control, n = 14; Speg-KO, n = 14; Speg-rescue, n = 14). Body weight of male (D) and female mice (E) was measured at different time points once per week and monitored until 32 weeks (for males: control, n = 14; Speg-KO, n = 6; Speg-rescue, n = 15; for females: control, n = 12; Speg-KO, n = 9; Speg-rescue, n = 10). The average body weight of male Speg-rescue mice begins to differentiate from that of Speg-KO mice at 13 weeks of age (*P ˂ 0.05; 1-way ANOVA with Tukey’s post hoc test) and remains higher afterward, while the average body weight of female Speg-KO and Speg-rescue mice is not significantly different at most of the time points.

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  Figure 4. DNM2 reduction improves mouse motor function of Speg-KO mice. (A) Representative mouse activity maps from each genotype of mice and the corresponding activity type distribution graphs at 2 months of age. A clear improvement in ability to explore the arena is observed in Speg-rescue mice as compared with Speg-KO mice. R, resting time; S, slow movement; F, fast movement. (B) Distribution of mouse resting time, slow movement, and fast movement. Although not statistically significant, (C) Speg-rescue mice travel a longer horizontal distance (P = 0.08) than Speg-KO mice and (D) exhibit increased hind limb rearing behavior (P = 0.07) compared with Speg-KO mice. Control, n = 23; Speg-KO, n = 6; Speg-rescue, n = 9. *P < 0.05; **P < 0.01; ***P < 0.001; 1-way ANOVA with Tukey’s post hoc test.

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  Figure 5. DNM2 reduction rescues skeletal muscle histology and improves contractility in extensor digitorum longus muscles of Speg-rescue mice. (A) Representative TA muscle images of control, Speg-KO, and Speg-rescue mice at 3 months of age. (B) H&E stains of TA muscles from control, Speg-KO, and Speg-rescue mice at 3 months of age. Scale bar: 100 μm. (C) Distribution of the cross-sectional area (CSA) in control, Speg-KO, and Speg-rescue TA muscles (n = 4 per group). (D) The mean CSA of Speg-rescue TA muscles is significantly larger than that of Speg-KO (****P < 0.0001, over 500 fibers were analyzed from each group; 1-way ANOVA with Tukey’s post hoc test). (E) Absolute peak tetanic force was significantly reduced in EDL muscles from Speg-KO mice compared with muscles from control animals. Muscles from the Speg-rescue mice were not different from either of the other groups. (F) Peak tetanic force expressed relative to EDL physiological CSA. Peak force/CSA was significantly reduced in Speg-KO animals (compared with control) but was restored to control levels in the Speg-rescue group. (G) Force-frequency relationships of EDL muscles. Forces obtained at different frequencies of stimulation…

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  Figure 6. DNM2 reduction restores the localization of triadic proteins, triad ultrastructure, and triad number in Speg-rescue skeletal muscle. (A) Transverse TA muscle sections stained for DHPRα1, RyR1, SERCA1, and DNM2. Abnormally accumulated DHPRα1 (denoted by arrowheads), SERCA1 (denoted by arrows), and DNM2 (denoted by asterisks) were evident in discrete areas of Speg-KO myofibers yet absent in Speg-rescue mice (over 100 fibers were analyzed from each group, n = 3 per genotype). Scale bar: 50 μm. (B) Electron micrographs in quadriceps specimens obtained from control, Speg-KO, and Speg-rescue mice at 3 months of age. The upper panel shows an overall organization of muscle structure, and the lower panel shows an enlarged view of triad ultrastructure (white arrows) from each group. Abnormal and fewer triads (white asterisk) are noted in Speg-KO mice. (C) The number of triads per 50 μm2 was significantly decreased in Speg-KO mice compared with control mice. However, the triad number in Speg-rescue mice was remarkably increased compared with Speg-KO mice. Each dot represents a randomly selected field to count the triad number; *P ˂ 0.05; ****P < 0.0001; n = 3 per genotype; 1-way ANOVA with…

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  Figure 7. Restoration of PI3P levels in the Speg-rescue mice. (A) Immunoblot and quantification of MTM1 expression in triceps (****P ˂ 0.0001, n = 3 per genotype; 1-way ANOVA with Tukey’s post hoc test). (B) PI3P levels are increased in Speg-KO muscle, while PI3P levels in Speg-rescue muscle are similar to control muscle, as determined using a PI3P ELISA kit [purified lipid (pmol)/mass (g) of quadriceps muscle]. PI3P levels in control muscle = 71.5 ± 15.1 pmol/g (n = 4), Speg-KO muscle = 166.5 ± 6.5 pmol/g (n = 4), and Speg-rescue muscle = 78.1 ± 10.8 pmol/g (n = 3). ****P < 0.0001; 1-way ANOVA with Tukey’s post hoc test. (C) Immunostaining for PI3P on TA muscle. Abnormal PI3P accumulation (denoted by arrows) was detected in discrete areas of Speg-KO myofibers yet absent in Speg-rescue mice. Scale bar: 50 μm; over 100 fibers were analyzed from each group, n ≥ 3 per genotype.